Materials/Equipment
Yeast
Glucose
Distilled water
Sodium hypochlorite solution (15% w/v)
Potassium iodide solution (10% w/v)
2 Conical flasks (250 cm3)
Graduated cylinder (250 cm3)
2 Graduated cylinders (50 cm3)
2 Beakers (250 cm3)
2 Beakers (50 cm3)
2 Boiling tubes
Thermometer Funnel | Filter paper
Timer
Labels
2 Syringes (5 cm3)
2 Rubber bungs
2 Fermentation locks
Electronic balance
Weigh boats
Incubator (25 °C 30 °C)
Water bath (50 °C 60 °C) |
Procedure
- Familiarise yourself with all procedures before starting.
To produce alcohol using yeast - Prepare 500 cm3of a 10% w/v glucose solution.
- Into each of the two conical flasks, add 250 cm3 of the 10% w/v glucose solution.
- To one, add 5 g of yeast and swirl. Label this `yeast + glucose solution'.
- The second flask acts as the control (has no yeast). Label as `control'.
- Attach a fermentation lock (half-filled with water) to each flask.
- Place both flasks in the incubator at 30 °C overnight.
To show the presence of alcohol: Iodoform test for alcohol - Remove both flasks from the incubator and filter the contents of each into separate beakers and label as before.
- Transfer 3 cm3of the yeast and glucose filtrate into a test tube and label.
- Transfer 3 cm3of the control filtrate into another test tube and label.
- To each test tube, add 3 cm3of the potassium iodide solution and 5 cm3of the sodium hypocholorite solution.
- Warm gently for 4 5 minutes in the water bath.
- Allow to cool and observe any change(s).
- Record and compare results.
- Replicate the investigation or cross reference your results with other groups.
Result
| Flask | Original colour
of filtrate | Final colour
of filtrate | Other change(s) |
| Yeast and glucose solution | | | |
| Control (no yeast) | | | |
Conclusion/Comment
SKILL ATTAINMENT
Following instructions
Familiarise yourself with all procedures before starting
Follow instructions step by step
Listen to the teacher's instructions
Correct manipulation of apparatus
Use the graduated cylinder to measure the volumes of glucose solution
Use the syringe for measurement of small volumes
Use the electronic balance
Attach the fermentation locks to the conical flasks
Filter the suspension
Set the incubator
Set and maintain the water bath
Use the timer
Observation
Observe bubbles of carbon dioxide being liberated
Observe the effect of filtering
Observe colour changes during the iodoform test
Observe the presence/absence of yellow crystals
Recording
Write up the procedure
Record any colour changes during the iodoform test
Record the presence/absence of yellow crystals
Interpretation
Draw reasonable conclusions from your observations and results
Application
Become aware of any other application(s) of what you learned in this activity
Organisation
Exercise caution for your personal safety and for the safety of others
Work in an organised and efficient manner
Label as appropriate
Work as part of a group or team
Clean up after the practical activity
Background information
Alcohol production by fermentation is a well established technology which has long been practised throughout the world. Continuous alcohol production with the use of immobilised yeasts, gives rise to more efficient fermentation.
Alcohol is produced by yeast fermentation of the sugars of various plants. Yeasts ferment simple sugars (monosaccharides) into carbon dioxide and ethanol under anaerobic conditions. Yeasts are single-celled fungi. The genus Saccharomyces is the one most commonly used due to its efficient alcohol production and tolerance of high alcohol levels. Some yeasts can live until the alcohol concentration reaches 18%.
Iodoform test
Alcohol groups when treated with potassium iodide (KI) and sodium hypochlorite (NaOCl) readily yield iodoform (CHI3).
There are three stages in this reaction: a)Oxidation: aqueous sodium hypochlorite oxidises the potassium iodide to potassium hypoiodite, this then oxidises the alcohol to an aldehyde. C2H5OH CH3CHO
ethanol ethanal
b) Substitution: potassium hypoiodite then iodinates the aldehyde to tri-iodoethanal (iodal). CH3CHO CI3CHO
ethanal iodal
c) Hydrolysis: aqueous sodium hypochlorite always contains sodium hydroxide, which converts the iodal to triiodomethane (iodoform) and sodium methanoate CI3CHO + NaOH CHI3 + HCOONa
iodal + sodium hydroxide iodoform + sodium methanoate
Formation of solid iodoform (yellow crystals) is a positive result. Iodoform is a disinfectant and can be used as an external antiseptic.
Advance preparation
- Set the incubator.
- Set the water bath.
- Check the temperatures of the incubator and water bath with a thermometer.
- Prepare the following solutions: glucose solution (10% w/v), sodium hypochlorite solution (15% w/v), potassium iodide solution (10% w/v).
Helpful hints
- Immobilised yeast may be used instead of dried yeast - this eliminates the need to filter.
- In the iodoform test the sodium hypochlorite used must contain some sodium hydroxide. Use commercial bleach and add sodium hydroxide if necessary.
- Use pure ethanol to observe positive result in the iodoform test.
- Use a cork borer when inserting tubing into bungs.
- Using long necked conical flasks and setting the incubator at 25 °C would help to prevent overflow during fermentation.
- A Bunsen valve can be used instead of a fermentation lock during fermentation.
To construct a Bunsen valve
a) Insert a short piece of glass tubing into a single holed bung.
b) Attach approximately 4 cm of rubber tubing to the glass tubing.
c) Using a scalpel blade, carefully cut a small vertical slit in the rubber tubing. This allows the gas to escape but will prevent air from entering the flask.
d) Use a Hoffman clip to seal the end of the rubber tubing (above the slit).